Lyophilization pretreatment facilitates extraction of soluble proteins and active enzymes from the oil-accumulating microalga Chlorella vulgaris

Microalgae are being developed as a renewable source of triacylglycerides (TAGs) for industrial production of biodiesel, bioplastics, and biolubricants. Certain species such as Chlorella vulgaris are attractive models for studies of microalgal TAG biosynthesis because they accumulate significant quantities of TAGs when cultured mixotrophically in the presence of exogenous carbon sources such as glycerol. As the major byproduct of biodiesel production from TAG, glycerol recycling by bio-oil accumulating microalgae would be particularly advantageous. However, our current understanding of the organization and control of microalgal glycerol uptake, metabolism, and incorporation into TAG is very limited. One reason is that microalgae such as C. vulgaris are endowed with a complex and robust cell wall that significantly impedes cellular lysis for efficient extraction of soluble proteins and active enzymes. The aim of the current study was to optimize extraction of soluble proteins and glycerol kinase activity from glycerol-supplemented, mixotrophic cultures of C. vulgaris. Lyophilizing harvested cells prior to lysis using a French press, bead beater, mortar and pestle, or ultrasonication resulted in large (up to 6-fold) increases in soluble protein concentration (up to 60 mg/gFW) and glycerol kinase activity (up to 2 mu mol/min gFW(-1)) in the resultant clarified extracts. In particular, the activity of glycerol kinase, hypothesized to catalyze a rate-determining step in the metabolism of exogenous glycerol into TAGs, was virtually undetectable in clarified extracts unless the harvested cells were lyophilized prior to extraction. This method was successfully applied to other key carbohydrate metabolizing enzymes (pyruvate kinase) and oleaginous microalgal species (Scenedesmus sp.). Deciphering the organization and control of microalgal carbon (including glycerol) metabolism is expected to facilitate harnessing the full potential of TAG-accumulating microalgae for downstream biofuel and industrial biotechnology applications.